Effect Of Resveratrol On MAPK Inflammatory Signal Transduction Pathway In Murine Macrophage Cell Line

[Background]As we all know, MAPK (Mitogen-Activated Protein Kinase) signal transduction pathway is a very important intracellular signal transduction pathway. MAPK pathway has three classic subfamily members ERK1/2 (extracellular signal-related kinases 1 and 2), JNK (c-Jun N-terminal kinase) and p38 MAPK (p38 Mitogen-Activated Protein Kinase). Produce a large number of inflammatory mediators, thus promoting the development of inflammation LPS and other factors can activate MAPK pathway. Activation of these pathways is characteristic with the increasing expression of their phosphorylated forms.Resveratrol exists widely in veratridine, cuspidatum, grape, peanut and other food and plant. Since resveratrol was initially found as a phytoalexin, its existing reliable and extensive anti-inflammatory activity has attracted more and more attention. Resveratrol can be used to treat corrosive esophagitis, acute pancreatitis, colitis and other inflammatory diseases, but its specific anti-inflammatory mechanisms are unknown. Since the MAPK pathway can promote the development of inflammation, we assumed that the anti-inflammatory mechanisms of resveratrol was related to the inhibition of MAPK pathway. To the best of our knowledge, studies addressing the relationship between anti-inflammatory mechanisms of resveratrol and MAPK pathway are limited. Our study is to investigate the effect of resveratrol on LPS-induced MAPK inflammation signal transduction pathway in RAW264.7.[Objective]To investigate the effect of resveratrol on LPS-induced MAPK inflammation signal transduction pathway in RAW264.7.[Methods](1) Effect of various concentrations of resveratrol on cell viability. Groups:①zero setting Group:DMEM medium②control group:untreated cells③treatment group:treating cells by resveratrol and LPS. Each group has five holes. Raw264.7 cells were seeded onto a 96-well culture plate at a density of 1×104 per well with 100μl of DMEM culture medium containing 10% FBS, 100U/ml penicillin, and 100μg/ml streptomycin and incubated for 24 hours at 37℃with 5% cox-2 atmosphere. Then add resveratrol and polysaccharide, so that the final concentrations of resveratrol in the medium were 5μmol/, 10μmol/1,15μmol/1,30μmol/1 respectively, and the final concentration of LPS is lμg/ml. After incubating for 24 hours, add 10ul of cck-8 solution, mix and incubate for 2 hours. Scan the plate with a microplate reader at absorbance 450nm department.(2) explore the time gradient of MAPK activation. The cells were seeded onto dishes and incubated at 37℃with 5% cox-2 atmosphere until confluence reached 80-90%. cells were taken out from the incubator. add LPS to culture and mix. Six different culture dishes were used to detect activation of MAPK at different time points. After treated, cells were washed with cold PBS and cell pellets lysed for 30 min with a lysis buffer followed by centrifugation for 5 min at 14,000g. Take the supernatant and add the loading buffer. Afterward protein concentrations were measured by BCA protein assay. Equal amount of the sample was seperated by SDS-PAGE. Then cut the gel, transfer it onto polyvinylidene fluoride membrane, block it, incubate it with primary antibody and secondary antibodies respectively and then detect signal sequentially.(3) to detect effect of various concentrations of resveratrol on the MAPK pathway. Experimental groups:①control group②treatment group by lps③treatment groups with LPS and resveratrol at different concentrations. Resveratrol added to the medium, so that the final concentration in medium, respectively, is 5μmol/l, 10μmol/l,15μmol/l. Other operations is the same as above.[Results](1) Effect of different concentrations of resveratrol on cell viability. CCK-8 was used to test effect of various concentrations of resveratrol on cell viability. We found, resveratrol ranging from 5 to 15μM has no effect on cell viability, when the concentration of resveratrol reached 30μM, the cells viability began to decline.(2) Activation of MAPK by LPS. In order to explore the dynamics of MAPK phosphorylation triggered by LPS. We detected the expression levels of the MAPK pathway proteins at six different time points by western blot. Phosphorylated forms of MAPK subfamily proteins increased at 5 minute peaked at 30 minute and declined 60 minute thereby.(3 Inhibitory effect of resveratrol on MAPK pathway. We tested the impact of different concentrations of resveratrol on MAPK activity. We found that resveratrol can inhibit phosphorylation of p38 mapk and JNK induced by LPS.[Conclusion]Resveratrol ranging from 5 to 15μM could inhibit the phosphorylation of p38 MAPK and JNK,but had no effect on phosphorylation of ERK1/2.